Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In this approach, genes that encode foreign antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Significant progress has been made in the development of attenuated Salmonella enterica serovar Typhi live vector vaccines. Advances have been made in genetically stabilized expression plasmids, antigen export systems to improve foreign antigen-specific immunity, and the establishment of intranasal models in both mice and non-human primates for characterizing mucosal, humoral, and cellular immune responses to these live vectors. Of critical importance to future clinical trials, non-antibiotic plasmid selection systems have recently been engineered to improve the safety of these vaccines. The broad hypothesis of this research plan is that by appropriate manipulation of the Salmonella enterica serovar Typhi live vector platform technologies, we can construct a mucosally administered multivalent vaccine to immunize against recurrent Clostridium difficile infections (RCDI). To accomplish this, we will engineer within a single strain both non-antibiotic plasmid-based and novel chromosomal expression systems to express in concert the non-toxic cell-binding domains of three C. difficile toxins; enterotoxins A and B and binary toxin. The immunogenicity of this live vector strain will be determined using a murine intranasal model of immunogenicity, and a vaccination strategy based on a heterologous prime/boost approach in which the host is immunized sequentially with C. difficile antigens delivered both using live vectors and as purified proteins. Promising candidates eliciting antibody responses against all three C. difficile antigens will be further tested for protection in mice similarly immunized with he multivalent strain and challenged orogastrically with spores from an epidemic strain of C. difficile.